A variety of techniques have been used to microdissect specific cells or cell populations from a histological sample under direct microscopic visualization. Original microdissection techniques involved painstaking (and sometimes clumsy) manual dissection using needles or other micro-manipulation devices to isolate individual cells based on visible, histological characteristics.
More recent techniques have been developed to more efficiently separate biological components, such as particular subsets of cells, from a whole tissue sample. For example, Emmert-Buck et al. described the use of laser-based microdissection techniques to rapidly procure microscopic, histopathologically defined cell populations. Examples of such laser capture microdissection (LCM) are shown in U.S. Pat. Nos. 5,843,657; 5,843,644; 5,859,699; 5,598,085, and 6,010,888, as well as WO 97/13838; WO 98/35216; WO 00/06992; and WO 00/49410. The disclosure of columns 4-18 of U.S. Pat. No. 6,010,888 is incorporated herein by reference. In LCM, a tissue section is contacted with a transfer member that is selectively and/or focally activated by an external force to adhere target cells to the activated region of the transfer member. For example, a laser beam can be directed in a microscopic field of view toward a portion of the transfer member that overlies the target cells. The laser beam focally activates the transfer member to adhere the target cells to it, and the transfer member is then pulled away from the tissue section to remove the adherent targeted cells from the tissue section for subsequent analysis.
Other microdissection techniques are disclosed in U.S. Pat. No. 6,194,157, which describes overlaying a photoresist (such as those used in etching computer chips) onto a thin tissue section, then activating specific regions of the photoresist using electromagnetic radiation (such as a beam of a laser). Depending on the photoresist used, the “desired” cells are either washed off in the activated areas, or the undesired cells are washed away while the activated photoresist holds the desired cells to the slide. These methods share the same inherent disadvantages of LCM, in that individual cells must be visually identified and targeted before harvest.
A more recent approach to the analysis of biological material is layered expression scanning (LES), as disclosed in WO 01/07915. A biological sample (such as a tissue section) is placed on a layered substrate, in which different layers contain different identification molecules, for example different monoclonal antibodies or nucleic acid probes. Components of the biological sample are then transferred through the layers, by diffusion or electrophoresis, such that different components of the specimen are specifically bound in different layers. The pattern of binding in the different layers can be correlated with the architecture of the biological specimen, to determine different patterns of molecular expression in different regions of the specimen. For example, differences in protein expression can be compared between regions of malignant and non-malignant cells in a heterogeneous tumor specimen.
Another recent advance in the field of microdissection is the transfer microdissection technique shown in WO 02/10751. The transfer of targeted specimen components is accomplished by selectively focally altering a characteristic of a transfer layer adjacent the target region, such that biomolecules can move through the altered area of the transfer layer. In particular examples, the transfer layer is altered by focally increasing a permeability of the transfer layer, for example by selecting and removing a focal portion of the transfer layer. The biomolecules are then transported through the altered region of the transfer layer, to microdissect the biomolecules of interest from the biological sample. Transfer microdissection allows biomolecules from regions of interest in the biological specimen (such as nests of highly atypical cells in a tumor section) to be selectively analyzed.
Although these microdissection techniques have provided powerful tools for the selective analysis of biological specimens, and are a substantial improvement over prior techniques, they are still not highly susceptible to automation for high-throughput analysis of multiple specimens. These techniques still rely on selection of a targeted structure, for example by microscopic visualization and manual targeting with a laser beam that is directed at the target to adhere it to an overlying thermoplastic film.